Assays to determine vitamin B12 levels in humans have long been recognized as a valuable tool in the diagnosis and treatment of such diseases as pernicious anaemia. Vitamin B12 is required for the maintenance of homeostasis.
Vitamin B12 was first purified in 1948. Its complete molecular structure was subsequently elucidated. It has an organometallic corrin nucleus with a stabilized central cobalt ion. The biological activity of such corrin compounds is determined by the composition of the side chain, of which only molecules possessing an “A” ring conjugated nucleotide alpha-ribazole side chain stabilizing the cobalt residue are active.
Intrinsic factor was first noted in 1929. The lack of intrinsic factor was found to result in the development of pernicious anemia. The polypeptide is present in gastric secretions and facilitates the physiological uptake of vitamin B12. Intrinsic factor is of 55–60 k molecular weight, consisting of a single continuous peptide strand configured with a single B12 binding site. The material is recognized for its high specificity to only biologically active forms of B12. Upon binding B12, intrinsic factor undergoes a conformational change which accommodates binding to the intrinsic factor receptor; this conformational change is associated with an increase in affinity toward the intrinsic factor receptor.
A second B12 binding protein found in gastric secretions is R protein. R protein lacks the stringent specificity of intrinsic factor. Specifically, R-protein binds all corrin ring structures including cobamide, cobinamide and cobrinamide. R-protein has been identified as a contaminant of intrinsic factor preparations, and as such, can adversely impact the apparent specificity of said preparations.
Vitamin B12 assays were initially microbiologic. Later, after the specificity, affinity and stability of intrinsic factor was recognized, radioassays were developed which involved a competition reaction between radiolabelled vitamin B12 and vitamin B12 in a sample for binding to intrinsic factor.
Studies have shown that autoantibodies of certain patients directly block binding of vitamin B12 to intrinsic factor. Autoantibody fractions have been identified. Type I autoantibodies block vitamin B12 binding, hence, interfering with the function of intrinsic factor. Accordingly, it has been suggested that such autoimmune antibody be used to confirm the specificity of vitamin B12 binding activity in intrinsic factor preparations. P. A. Villanova, National Committee For Laboratory Standards, “Guidelines For Evaluating a B12 (Cobalamin) Assay” (1980).
Monoclonal antibodies have recently been described with specificity to intrinsic factor, but not for diagnostic purposes. Smolka and Donaldson, Gastroenterology 98: 607–614 (1990). Using human intrinsic factor, Smolka and Donaldson raised murine hybridomas, which were identified by dotblot analysis against immobilized intrinsic factor, followed by reselection for the ability to facilitate the immunoprecipitation of intrinsic factor-vitamin B12 complexes. They then recloned positive clones. Some of the positive clones secreted antibodies which bind to intrinsic factor-B12 complexes.
Pourfarzaneh et al. (WO 91/00519) describe immunoassays for vitamin B12 involving monoclonal antibodies said to be specific to the intrinsic factor:vitamin B12 complex and to the vitamin B12 binding site on intrinsic factor. Immunoassays using an antibody specific for the B12 binding site of intrinsic factor, such as the assays described in their application, require that the affinity and kinetics of the binding of the antibody to the B12 binding site be precisely balanced with the affinity and kinetics of the binding of B12 itself in order to obtain a assay of sufficient sensitivity. The kinetics of the interaction of B12 and intrinsic factor is slowed by the necessity of dissociating a particular antibody-intrinsic factor complex before B12 can bind to that intrinsic factor molecule and competitive assays of this type are slow and lack sensitivity.
Poufarzaneh et al also describe antibodies which are specific for the complex of intrinsic factor and vitamin B12. Such antibodies presumably are specific for epitopes which are made available by the conformational changes which occur in intrinsic factor upon B12 binding. Other researchers, as exemplified by Pfund et al., Molec. Immunol., 27:495–502 (1990), have described the use of conformation sensitive antibodies to detect conformational changes in a protein, and to detect conformation sensitive antibodies. However, immunoassays in which an antibody is allowed to bind to an analyte and the extent to which it is subsequently released by later conformational changes in the analyte determined have not been described.
Sensitive, rapid assays for vitamin B12 are desirable. Most of the immunoassays now used require the use of radioisotopes and require the use of R-factor free intrinsic factor or the use of vitamin B12 analogues which bind to the R-factor to increase the sensitivity and specificity of the assays. Such radioassays are described by way of example in U.S. Pat. Nos. 4,188,189 and 4,426,455.